The goal of this proposal is to conduct a comprehensive examination of the effect of aging on the mitochondrial proteome of mice. We have chosen to focus on the mitochondrial proteome for two reasons. First, there is almost unanimous agreement that mitochondria are very likely to play an important role in aging, either through changes in function (e.g., reduced energy generation or increase apoptosis) or through the generation of ROS, which in turn damage molecules leading to aging. Second, the mitochondrial proteome has a manageable number of proteins (1000 to 2000) to study using the current technology in proteomics. Although there are substantial data on the deterioration of mitochondrial function with aging, there is little information on the molecular basis of this phenomenon. We hypothesize that alterations in the mitochondrial proteome have a high probability of playing a major role in the age-related changes in mitochondria function. We proposed to test this hypothesis by performing the first detailed study of global expression and post-translational modifications in mitochondria proteome as a function of age by studying mitochondria isolated from various tissues of mice over their life span and determining the effect of dietary restriction (DR), which retards aging, on the age-related changes observed in the mitochondrial proteome. The Specific Aims of this project are to profile the effects of age and DR on 1) protein expression in the mitochondria proteome, 2) phosphorylation of the mitochondria phosphoproteome, 3) oxidative modification of mitochondria proteins, and 4) mitochondrial function. These studies will be conducted with a series of new proteomics methods we have developed specifically for the examination of protein expression, enzymatic post-translational modifications, and protein oxidation. Recognizing that different proteins are often seen with multidimensional chromatography and gel electrophoresis, we have designed methods that allow us to employ both techniques in our studies of the mitochondrial proteome.